Apparatus and method for nucleic acid isolation using supercritical fluids

ABSTRACT

A method for detecting the presence of a microorganism in an environmental sample involves contacting the sample with a supercritical fluid to isolate nucleic acid from the microorganism, then detecting the presence of a particular sequence within the isolated nucleic acid. The nucleic acid may optionally be subjected to further purification.

This application is a divisional of U.S. patent application Ser. No.08/733,816 filed Oct. 18, 1996 now U.S. Pat. No. 5,922,536.

FIELD OF THE INVENTION

The present invention relates to an apparatus and method for theisolation of nucleic acids from microorganisms. More particularly, theinvention relates to an apparatus and supercritical fluid method for theisolation of nucleic acids suitable for hybridization and/oramplification by the polymerase chain reaction.

BACKGROUND OF THE INVENTION

Since 1914, the safety of our water supply has generally been protectedby the use of assays which detect the growth of certain types ofbacteria, commonly referred to as indicator bacteria, to infer thepresence of pathogens. Indicator bacteria, such as fecal coliforms andtotal coliforms, are found in fecal matter along with many pathogens.Although many indicator bacteria do not cause disease in humans, theirpresence indicates a potential risk of exposure to pathogens. As aresult, water in which such bacteria are found will be declared unsafefor human contact therewith. Pathogens are not routinely assayed bydirect methods due to difficulty in their isolation and detection. Incontrast, indicator bacteria are cultured in 24 to 48 hours and can bedetected visually The main disadvantage with this detection method isthat the results do not indicate the present water quality. In addition,indicator assays fail to accurately assess the infectivity of water. Therisk is overestimated in environments which stimulate the growth of thenon-pathogenic indicator microorganisms (i.e. warm, nutrient-richwaters). Conversely, certain waterborne pathogens (i.e. Legionella andNaegleria fowleri) are not transmitted through the feces and thus arenot associated with fecal indicator organisms. Even in situations inwhich pathogens and indicator bacteria are from the same fecal source,the indicator bacteria may be killed more quickly than hardier pathogenssuch as protozoan cysts, viruses or bacterial spores. These and otherproblems could be eliminated by using assays which directly detect andquantitate waterborne pathogens.

More recently, the polymerase chain reaction (PCR), has been used in thedetection and classification of various microorganisms. While DNAhybridization is useful in some applications, it carries the distinctdisadvantage of having a high detection limit (low sensitivity). PCR, onthe other hand, eliminates the need to culture the microorganism and isextremely sensitive--capable of detecting a single cell. The first andmost critical step in both methods is, of course, the isolation of DNAof sufficient purity for analysis.

Several methods exist for the isolation of DNA from bacterial cells.These methods essentially utilize the same basic procedure. Bacterialcells are lysed enzymatically (i.e., lysozyme treatment), mechanically(i.e., bead homogenization) or by repeated freeze-thaw cycles, orcombinations of these, followed by dissolution of the cell membrane withalkali and detergents such as sodium dodecyl sulfate (SDS) (Maniatis etal., 1989; Tsai et al., Appl. Environ. Microbiol., 57:1070-1074, 1991;Bej et al., Appl. Environ, Microbiol., 57:1013-1017, 1991). The celllysate is then treated with proteinases and hexadecyltrimethyl ammoniumbromide (CTAB) to degrade proteins and precipitate carbohydrates,respectively. The most common proteinase used in this procedure isproteinase K. Finally, DNA is purified by extraction with phenol,chloroform and isoamyl alcohol. Variations of this basic method havebeen used to isolate DNA from soils, sediments and water samples for usein hybridization and PCR analysis (Somerville et al., Appl. Environ.Microbiol., 55, 548-554, 1989; Tsai et al., Appl. Environ. Microbiol.,59:353-357, 1993; Bej et al., Appl. Environ. Microbiol., 56:307-314,1990). Although these methods can result in DNA of sufficient purity forboth hybridization and PCR analysis, they are time consuming and involveexpensive and toxic reagents. Further, the DNA obtained from soil andsediment samples is often of questionable purity and its analysisrequires several days.

A substance in a supercritical fluid state is defined when it is abovethe critical temperature (the temperature above which the gas cannot beliquified no matter how high the pressure), and above the criticalpressure (the pressure which will liquefy the gas at its criticaltemperature). At this point, the fluid has equal coexisting densities ofits gaseous and liquid phases (Lange's Handbook of Chemistry, 13th ed,Dean, J. A., ed., McGraw-Hill, New York). The supercritical fluid is aviscous gas with properties analogous to those of liquid solvents(Hawthorne, Anal. Chem., 62:633-642, 1990). The difference betweenliquid solvents and supercritical fluids is that the solvent strength ofa supercritical fluid can be controlled by changes in temperature and/orpressure. The most commonly used supercritical fluid is carbon dioxide(CO₂) which is inert, nontoxic, nonflammable, inexpensive and availablein a very pure form. CO₂ has a low critical temperature (31.1° C.) andcritical pressure (72.85 atm).

Supercritical CO₂ has been used to extract a variety of nonpolarcompounds from both biological and non-biological sources (Lin et al.,Biotechnol. Prog., 8:458-461, 1992). It has been used to extractalkanes, sulfur compounds, PCBs, pesticides and polycyclic aromatichydrocarbons from soil and sediments (Hawthorne, ibid.; Hopfgartner etal., Org. Geochem., 15:397-402, 1990), as well as fatty acid and sterollipid biomarkers from plant tissue, sediments, and filtered watersamples (Klink et al., Org. Geochem., 21:437-441, 1994).

The number of viable microorganisms decreases after treatment withsupercritical fluids. For example, cell inactivation of Saccharomycescerevisiae increases with an increase in pressure at temperatures of25-45° C. and pressures of 68-204 atm (Lin et al., ibid.). Under theseconditions, inactivation occurred in greater than 15 minutes at 25° C.and 5 minutes at 35° C. Increases in pressure or exposure time werecorrelated with an increase in adverse effects, including microbialdeath (Hoover et al., Food Technol., 43:99-107, 1989). When exposed topressures of 300 to 450 atm, Pseudomonas exhibited morphological changesincluding cellular elongation, separation of the cell wall from theplasma membrane and clear areas of spongy or reticular structures in thecytoplasm (Hoover et al., ibid.; Kriss et al., Mikrobiolgiya, 38:88,1969).

DNA appears very resistant to hydrostatic pressure. Structural integrityof calf thymus or salmon sperm DNA remained unchanged when pressures ofup to 10,000 atm were applied for 60 min at 25-40° C.

The present invention provides an apparatus and method for the rapidisolation of DNA of high purity from microorganisms present inenvironmental samples including water, soil and sediments. Importantly,this method can be used to detect the presence of pathogenicmicroorganisms in water supplies.

SUMMARY OF THE INVENTION

One embodiment of the present invention is a method of identifying amicroorganism in an environmental sample, comprising contacting thesample with a supercritical fluid to extract DNA therefrom; anddetecting the presence of a nucleic acid sequence specific to themicroorganism. Preferably, the microorganism is a bacterium.Alternatively, the microorganism is a protozoan, parasite or virus.Advantageously, the bacterium is E. coli. According to one aspect ofthis preferred embodiment, the sample is water, soil or sediment.Preferably, the supercritical fluid is supercritical CO₂. The method mayfurther comprise applying the sample to a filter prior to the contactingstep. In another aspect of this preferred embodiment, the detecting stepis PCR or hybridization analysis. Preferably, the nucleic acid is DNA.Alternatively, the nucleic acid is RNA. The method may further compriseextracting the sample with one or more solvents or mixtures prior to thedetecting step.

Another embodiment of the invention is a subassembly for extracting andpurifying nucleic acid from microorganisms present in a sample,comprising:

a sample cartridge comprising a filter for receiving said sample; and

a collection cartridge comprising a matrix which binds nucleic acidfluidly connected to the sample cartridge.

Preferably, the filter is a bed filter. Advantageously, the subassemblyis disposable. In one aspect of this preferred embodiment, the nucleicacid is DNA. Alternatively, the nucleic acid is RNA. Preferably, thesample is water, soil or sediment and the matrix is hydroxyapatite.

Another embodiment of the invention is an apparatus for isolating andextracting nucleic acid from microorganisms contained within anenvironmental sample, comprising:

a sampling cartridge having an input end and an output end;

a collection cartridge connected to the sampling cartridge at the outputend, wherein the collection and sampling cartridges are surrounded by ahigh pressure compartment within a temperature- and pressure-controlledzone;

a high pressure interface sealingly engaging the input end of the samplecartridge;

a plurality of pumps fluidly connected to the sampling cartridge; and

a controller electrically connected to one of the pumps.

Preferably, the nucleic acid is DNA. Alternatively, the nucleic acid isRNA. According to one aspect of this preferred embodiment, the pumpspump sample, supercritical fluid, organic solvents, or aqueous buffer.Advantageously, the sampling and collection cartridges are disposable.Preferably, the sampling and collection cartridges are made of plastic.In addition, the pressure- and temperature-controlled zone may comprisean aluminum block.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing the percentage of E. coli and RhodococcusDNA recovered after supercritical CO₂ extraction under variousconditions.

FIG. 2 is a schematic diagram showing the disk sampling cartridge andcollection cartridge subassembly.

FIG. 3 is a schematic diagram of a bed filtration cartridge for use inthe sampling cartridge and collection cartridge subassembly.

FIG. 4 is a schematic diagram of an integrated nucleic acid extractionand purification system.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention includes the observation that intact genomic DNAcan be isolated by lysis of microbial cells with supercritical fluids.The method comprises exposure of microorganisms to supercritical fluids,resulting in cell lysis. Although the use of supercritical CO₂ ispreferred, the use of other supercritical fluids including propane,sulfur hexafluoride, Freons, nitrous oxide and ammonia is alsocontemplated. Although the DNA obtained by this method can be useddirectly for hybridization analysis or PCR, it is preferably furtherpurified by extraction with organic solvents (i.e., phenol, chloroform,isoamyl alcohol), polar solvents (i.e., ethanol, water), or combinationsthereof. The DNA may also be treated with proteinases (i.e. proteinaseK) to degrade proteins and with hexadecyltrimethyl ammonium bromide(CTAB) to precipitate carbohydrates.

The microorganisms which are detected are present in environmentalsamples, including water, sediments and soils. For detection ofmicroorganisms in water samples, the water is passed through a filterand the microorganisms are retained on the filter. The microorganismsare lysed directly on the filter. Suitable filters include, for example,membranes, glass fiber filters and bed filtration devices. For soil andsediment samples, microorganisms may be separated from the sample priorto supercritical fluid isolation, or the microorganisms in the samplemay be lysed directly, followed by removal of the nucleic acids from thesoil or sediment. Extraction and purification of microbial DNA fromsediments is discussed by Ogram et al. (J. Microbiol. Meth., 7:57-66,1987). The preferred method, direct lysis, may also comprise beadhomogenization (More et al., Appl. Environ. Microbiol., 60:1572-1580,1994). Pretreatment of the samples with organic solvents is alsocontemplated, as this procedure may facilitate cell wall degradation,thus increasing recovery.

A major advantage of the claimed nucleic acid isolation method is thatit is universal for all microorganisms. A significant disadvantage ofprior art lysis methods is that not all microorganism species are lysedby a particular procedure; in contrast, the instant method results inlysis of all species tested. This is important because if partial orpreferential lysis occurs, the extracted DNA would not be representativeof the population of microorganisms in the sample. The present method israpid and results in the isolation of high-quality DNA. Anotheradvantage of supercritical fluids, particularly CO₂, is that afterlysis, the fluid is removed from the sample in the form of a gasreducing the risk of sample loss due to pipetting or some other transfertechnique. Essentially, the lytic agent removes itself from the lysaterather than removing the lysate from the lytic agent. The supercriticalproperties of CO₂ thus eliminate the need for SDS as a lytic reagent.These current art procedures not only result in loss of sample, but insome cases are environmentally and physically unsafe.

Supercritical fluid may also be used to isolate RNA, including ribosomalRNA (rRNA) and messenger RNA (mRNA). Because of the ubiquitous presenceof RNases, cell lysis and extraction must be performed in the presenceof RNase inhibitors (i.e. RNasin) and all solutions should be made withdiethyl pyrocarbonate (DEPC)-treated water. Additionally oralternatively, microbial or environmental samples are extracted with hotphenol/chloroform at 60° C. which reduces RNA degradation due toinactivation of endogenous and exogenous RNase. The RNA obtained by thismethod can be used for Northern hybridization analysis,reverse-transcription, in vitro translation and for constructing cDNAlibraries. In contrast to DNA, RNA is not amplified directly, but isfirst transcribed into DNA by reverse transcriptase followed byamplification of the DNA. The detection of RNA is advantageous becausemultiple copies of the target RNA molecule may be present in the celland some forms of RNA (i.e. mRNA) rapidly degrade after cell death.Rapid degradation of the target nucleic acid is important if definitiveproof, of viability or infectivity is to be demonstrated because DNA maypersist for some time after cell death, resulting in detection of DNAfrom a nonviable pathogen.

DNA extracted from different environmental samples can be compared ifthey are normalized to the relative number of organisms originallypresent in the sample from which the total DNA was extracted. Typically,this normalization is performed by probing for the presence of 16S rRNAgene sequences in total DNA. A universal oligonucleotide probecomplementary to the 16S rRNA gene of eubacteria is incubated with theextracted DNA blotted onto a nylon membrane (Stahl et al., Appl.Environ. Microbiol., 54:1079-1084, 1988). The ratio between thefrequency of a specific gene and the sequences hybridizing to theeubacterial probe in total DNA is then determined. In addition, if it isassumed that there are about 5 copies of 16S rRNA genes per eubacterialcell, the hybridization can be used to estimate the overall number ofeubacteria in the sample.

An automated apparatus is used for extracting and purifying both RNA andDNA from microorganisms retained by filter systems such as filter bedsor membrane filters. The nucleic acid extracts are suitable for analysisby both conventional and state-of-the-art detection techniques. Solutes,including phospholipids, steroids and pollutants in the supercriticalfluid effluent and/or solvent extract can also be collected and analyzedby other conventional analytical techniques to provide complementarydata to facilitate sample characterization. The apparatus preparesnucleic acids more rapidly and less expensively than standard methodsallowing a more rapid detection of microorganisms contained in watersamples, including waterborne pathogens such as E. coli, Shigella,Cryptosporidium and Giardia. The apparatus can be used to detectmicroorganisms present in recreational waters, source water and potablewater.

In a preferred embodiment, the apparatus comprises a computerizedcontroller, pumps, a nucleic acid isolation module and a preferablydisposable subassembly containing a sample cartridge and a collectioncartridge. The nucleic acid isolation device comprises a temperature-and pressure-controlled zone, manifolds with automated valves,high-pressure fluid seals to receive the subassembly and a computerinterface board. In another preferred embodiment, the sample cartridgealso serves as a filtration device. The disposable subassembly protectsagainst cross-contamination. The first step, filtration, traps pathogenscontained in the sample on a filter system such as a commercial diskfilter (FIG. 2) or a filter bed housed in the sample cartridge (FIG. 3).

Disk filters have well-defined pore sizes (0.1 μm to 1 μm), goodtrapping efficiency and minimal dead volume which is important inminimizing the volume of solvent used for lysis, purification and samplecollection. Filters of different pore sizes may be used in combinationto maximize microorganism trapping efficiency. Filtration may beperformed using the sampling cartridge as a filter holder or by using auser-supplied filtering apparatus.

FIG. 2 illustrates one embodiment of the subassembly 2 of the invention.

The subassembly 2 contains a disk sampling cartridge 4 and a collectioncartridge 6 for use in isolation of nucleic acids from environmentalsamples. When the sampling cartridge 4 is used as the filteringapparatus, the water sample passes through inlet 8, upper porous frit 10and onto filter 12 within extraction chamber 14. Filtered samples passthrough filter 12 and lower porous frit 16. Porous frits 10 and 16 areattached to upper module housing 18 and lower module housing 20,respectively. If a user-supplied filtering apparatus is used, the filterwith trapped sample is placed in the sample cartridge and the cartridgesealed before nucleic acid isolation. The analysis of other samplesincluding urine, sputum, blood plasma, activated sludge and fermentationbroth, by this system is also contemplated. After filtration, thesampling cartridge 4 is fluidly sealed to collection cartridge 6 eitherby connector 22 or by an integral sealing system. The subassembly isthen sealed within the temperature- and pressure-controlled zone of thenucleic acid isolation module (FIG. 4).

The subassembly 2 is pressurized with carbon dioxide and heated abovethe critical point (73 atm, 31° C.) to form a supercritical fluid. Anexit valve is then opened to rapidly depressurize the system and rupturethe microorganisms within the sample cartridge. Hot organic solvent,(i.e. phenol, ethanol, isopropanol) is then pumped through inlet 8 usingstandard commercially available pumps (FIG. 4) into the system to: 1)further degrade the cell matrix; 2) deactivate enzymes which degradenucleic acids; and 3) remove contaminants.

Collection cartridge 6 is packed with a material 24 which has anaffinity for nucleic acids. Such materials include hydroxyapatite,silica and ion-exchange resin. Aqueous buffer is then pumped throughinlet 8 to transfer the nucleic acids from disk sampling cartridge 4 tocollection cartridge 6. The trapped nucleic acids are removed frommaterial 24 using appropriate buffers. For example, trapped nucleicacids are removed from the hydroxyapatite material 24 using 0.5 M sodiumphosphate, pH 6.8. The eluted nucleic acids pass through outlet conduit26 which is fluidly connected to cartridge 6, then analyzed byconventional techniques, including hybridization analysis, dot blotanalysis and PCR. Detection kits containing the probes for specificpathogens may be prepared and used with the system. For example, probescan be selected for their ability to determine specific sources of fecalcontamination in recreational waters. All reagents are provided in akit, including filter cartridges, labeled oligonucleotides, PCR reagentsand internal controls.

FIG. 3 shows an alternate sample cartridge configuration, the bedfiltration cartridge 30 for use in the subassembly 2 shown in FIG. 2.Bed filtration cartridge 30 contains inlet 32 attached to cap 34. Cap 34is threadedly sealed to cartridge housing 36 containing filter bed media38 within extraction chamber 40. Porous frits 42 are situated insidechamber 40 above and below filter bed media 38. The bottom of chamber 40is connected to the remainder of the subassembly by connector 22 asshown in FIG. 2. In this embodiment, the filter bed media 38 can bereplaced with other particulate matter such as soil or sediment foranalysis of the nucleic acids contained therein.

The complete automated nucleic acid purification and extraction system(NEPS) 50 is shown in FIG. 4. Parameters including pressure,temperature, composition of the supercritical fluid (modified or purecarbon dioxide) and solvent, exposure and extraction times and flowrates are set and monitored by controller 52 to optimize extractionefficiency. The system comprises a computerized controller 52, aplurality of pumps 54, and a nucleic acid isolation module 56. Thenucleic acid isolation module 56 is equipped with inlet 58 and outlet 60fluid manifolds, inlet seals 62, outlet seals 64, a temperature controlblock 66, a high pressure compartment 68 formed with the inlet sealingdevice 70 and the subassembly 2 containing the sampling 4 and collection6 cartridges.

In a preferred embodiment, the temperature control block 66 is made ofaluminum and heated by heating elements and, if needed, cooled with aPeltier device. In another preferred embodiment, the subassembly 2 isconstructed of materials capable of withstanding high-pressure operationsuch as aluminum or steel, and sealed by the inlet sealing device 70 tothe inlet manifold 58. In this embodiment, only the interior of thesubassembly 2 is pressurized.

Samples are applied onto either filter beds or commercial disk filtershoused within sample cartridge 4 which is preferably made of plastic.Alternatively, bed sampling cartridge 30 may be used in the NEPS.Application of samples is accomplished by vacuum filtration or use ofhead pressure to force aqueous solutions through the bed sampling 30 ordisk 4 sampling cartridge. Optionally, aqueous solutions may bepretreated with flocculation or coagulation agents to increasefiltration efficiency. Once a desired volume of liquid is filtered, thecolumn is drained and detachable endcaps are affixed to minimize therisk of contamination. After filtration, the endcaps are removed fromsample cartridge 30 and the nucleic acid collection cartridge 6 isconnected to the sample cartridge 30 using connector 22. The nucleicacid collection cartridge is preferably made of plastic. Thesample/collection cartridge subassembly is fluidly connected to an inletsealing device 70 and sealed into the temperature- andpressure-controlled zone 72 of the NEPS. In a preferred embodiment, thisstep is performed automatically, directed by the controller 52.Alternatively, the connection and sealing may be done manually. Oncesealed, the NEPS is pressurized with carbon dioxide via pump 54A whichis fluidly connected to sample cartridge 30 through conduit 74, manifold58 and conduit 76. The temperature control block 66 is sealed with ahigh-pressure interface to form the high pressure compartment 68 whichcontains the subassembly 2. To ensure that the pressure differentialdoes not become great enough to damage the sample/collection cartridgesubassembly 2, the pressure inside and outside the subassembly 2 ismonitored during pressurization. External pressurization is accomplishedby opening an automated valve within inlet manifold 58 which is in fluidcommunication with external pressurization conduit 78. Fluids areremoved from the NEPS via outlet conduit 80.

The subassembly is also heated to a predetermined temperature. before,after or simultaneously with pressurization. The protocol and setpointsfor pressurization and heating can be determined using routine protocolswell known to one of ordinary skill in the art to find those whichmaximize lysis efficiency for a pathogen of interest. Once pressurizedand heated, the system is equilibrated and the sample is exposed to thesupercritical fluid for a set period of time. Pump 54A pumpssupercritical fluid through conduit 74, inlet manifold 58 and conduit 76into sampling cartridge 30. After the temperature and pressure areraised above the critical point, then the liquid becomes supercritical.After a given period of time to allow supercritical fluid entry into thecells, typically several minutes, an exit valve is opened to rapidlydepressurize the system. The process may be repeated to increase lysisefficiency. Supercritical fluid treatment is performed in either astatic (no flow) or dynamic mode (i.e. flow rate of 1.5 ml/min). Dynamictreatment comprises opening an automated valve on the outlet manifold 60to cause flow through a restrictor. At the end on the exposure time, thevalve can be closed and the outlet valve opened for rapiddepressurization. The controller 52 directs all valve openings andclosings (manifold 58 and outlet conduit 80), as well as monitor andcontrol temperature and pressure.

If required, hot organic solvent(s) is pumped with pump 54B throughconduit 82, manifold 58 and conduit 76 into bed sampling cartridge 30 tofurther degrade the cell matrix; deactivate enzymes which can degradenucleic acids; and remove contaminants. The temperature and pressure areagain set and monitored by the controller. Carbon dioxide may bereintroduced into the system to remove any residual solvents. Aqueousbuffer is pumped through sample cartridge 30 using pump 54C which isfluidly connected thereto via conduit 84, manifold 58 and conduit 76, totransfer the extracted nucleic acids to the collection cartridge 6. Thesubassembly 2 is removed from the NEPS and the collection cartridge 6separated from the sampling cartridge 30. The trapped nucleic acids areeluted from the collection cartridge and amplified using conventionaltechniques, preferably PCR. In addition, the sample may be directlyeluted into a PCR module to further avoid cross-contamination.

This apparatus bridges the gap between the environmental sample andmodern molecular techniques such as PCR, thus providing direct evidenceof the presence of pathogens within about 6 hours rather than days at alow cost. Solid phase collection columns may be used to collect,concentrate and purify the extracted DNA and RNA, thus preventingcontamination thereof. Solid phases such as silica, hydroxyapatite,ion-exchange resin and other commercially available materials can beused.

In a preferred embodiment, due to the extremely high sensitivity of thePCR assay, the module for holding the filters during supercritical fluidisolation is disposable to eliminate cross contamination. Plasticextraction chambers can be used if both the inside and outside of themodule are pressurized at the same time. Alternatively, plastic chambersmay be inserted into high-pressure vessels to avoid damage to thechambers. The extraction chambers may be constructed of, for example,polyetheretherketone (PEEK), polyethylene, polypropylene and compositeglass. The filter module may be of two general shapes: a cylindricalform packed with particular filter media, and a flat form using amembrane or filter as shown in FIG. 2. The flat form is particularlyuseful because it minimizes the dead space and the surface area ofsample matrix. The modules may also be multi-use and constructed ofmetals such as stainless steel or aluminum.

Samples of various bacterial species were lysed using supercriticalfluid as described in the following example.

EXAMPLE 1 Sample Preparation and Supercritical Fluid Treatment

Cultures of E. coli were grown in LB broth (10 g Bacto-Tryptone, 5 gyeast extract, 10 g NaCl per liter) to 35° C. to an optical density (OD)of 1.0 at 546 nm. Cultures of Pseudomonas fluorescens HK44 (King et al.,Science, 249:778-781, 1990), Sphingomonas paucimoblis A8AN,Mycobacterium (Wang et al., Environ. Sci. Technol., 30:307-311, 1996)and Rhodococcus sp. SM1 (Malachowsky et al., Appl. Environ. Microbiol.,60:542-546, 1994) were grown in YEPG broth (1 g dextrose, 2 gpolypeptone, 0.2 g yeast extract, 0.2 g NH₄ NO₃ per liter) at 27° C. toan OD₅₄₆ of 1.0. Cells were collected by filtration of 1 ml culturethrough sterile 25 mm GF/F glass fiber filters (Whatman, Hillsboro,Oreg.). Bacterial concentration was determined by dilution plating andstaining with acridine orange (Atlas et al., Experimental Microbiology:fundamentals and applications, second edition, MacMillan Publishing Co.,New York, 1988).

SFE grade CO₂ was used as the supercritical fluid in an Isco SFX2-10supercritical fluid extractor equipped with two 260D syringe pumps. Thefilters containing microorganisms were folded twice and clipped on theunfolded edge with a paper clip to avoid loss of cells. The samples werethen placed in an extraction chamber and heated to the desiredtemperature. After 1 minute, the chamber was pressurized to the desiredpressure. The restrictor valve was opened, and the fluid flowed throughthe extraction chamber at a rate of 1.5 ml/min. At the end of the runtime, the outlet valve was opened and the inside pressure was rapidlyreduced to atmospheric pressure. The filter was then removed forprocessing.

EXAMPLE 2 Supercritical CO₂ Lysis Efficiency

Glass fiber filters containing cells lysed by supercritical CO₂treatment were placed in microcentrifuge tubes to which a known volumeof TE buffer had been added. The samples were vortexed for 20-30 secondsand a 10 μl aliquot was removed, applied to a 25 mm polycarbonate filter(0.2 μm pore size, Poretics, Livermore, Calif.) and stained withacridine orange. A serial dilution series was also prepared forinoculation on YEPG agar plates. The cell count determined by acridineorange staining after supercritical CO₂ treatment reflects the number ofintact bacteria, while dilution plating represents the number of viablecells. The number of intact bacteria after treatment and the originalbacterial concentration were used to calculate lysis efficiency. Lysisefficiency of E. coli was originally tested at different exposure times,pressures and temperatures and ranged from 74% to 97%. In general,favorable lysis was obtained at all conditions tested.

Similar experiments were performed at 80° C. and 400 atm using speciesof Pseudomonas, Sphingomonas, Mycobacterium and Rhodococcus. Resultsfrom lysis of these species under supercritical conditions were comparedto those obtained using a conventional SDS lysis procedure (Table 1).For Pseudomonas, Sphingomonas and E. coli, the lytic action ofsupercritical fluids is comparable to that of SDS and, in the case ofRhodococcus and Mycobacterium, supercritical fluids are a better lyticagent. Dilution plate counts of lysed bacteria showed no growth aftertreatment with SDS or exposure to supercritical conditions, indicatingthat both methods result in loss of bacterial viability.

                  TABLE 1                                                         ______________________________________                                                % SFE Lysis       % SDS Lysis                                                 by AODC Standard  by AODC   Standard                                          count   Deviation count     Deviation                                 ______________________________________                                        E. coli   88.83     2.98      90.23   1.85                                    Pseudomonas                                                                             90.31     6.88      94.09   8.39                                    Sphingomonas                                                                            68.61     22.39     99.32   0.65                                    Rhodococcus                                                                             74.11     8.28      2.85    50.5                                    Mycobacterium                                                                           78.88     6.87      59.03   19.78                                   ______________________________________                                    

EXAMPLE 3 Recovery of DNA From Glass Fiber Filters

To determine a suitable buffer for recovery of DNA from glass fiberfilters, ³² P-labeled plasmid DNA was applied to the filters. Thefilters were placed in tubes containing either TE buffer (10 mMTris-HCl, pH 8.0, 1 mM EDTA) or 0.12 M Na₂ HPO₄. pH 8.0. The tubes wereshaken for several minutes and an aliquot was placed in a scintillationvial to determine the amount of radiolabel released from the filters. TEbuffer recovered 84.2±4.8% of the radiolabeled DNA while PO₄ bufferrecovered 57.3±1.5% of the DNA.

EXAMPLE 4 Integrity of DNA After Exposure to Supercritical CO₂

To determine the integrity of DNA after exposure to supercriticalconditions, the TA cloning vector pCRII (Invitrogen, San Diego, Calif.)containing a 1.1 kb DNA insert was pipetted onto GF/F filters andexposed to supercritical conditions. The time of exposure varied fromrapid pressurization-depressurization to 30 minutes in increments of 10minutes. Experimental controls consisted of DNA which was placed on thefilter, but was not exposed to supercritical conditions.

After exposure to supercritical conditions, the filters were placed in1.5 ml Eppendorf tubes to which 750 μl TE buffer was added. The tubeswere vortexed for 20-30 seconds. With the filter remaining in the tube,an equal volume of chloroform/isoamyl alcohol (24:1) was added to thetube and centrifuged for 5-6 minutes. This step was required due tobreakdown of the glass fiber filters into particles which interferedwith DNA recovery. This step allows removal of the glass particles whichstay in the lower organic layer, while the DNA is in the upper aqueouslayer. The aqueous layer, minus the filter, was removed and placed in anew tube. An equal volume of phenol/chloroform/isoamyl alcohol (25:24:1)was added and the samples were centrifuged for 5-6 minutes. The upperaqueous layer was recovered and precipitated with 0.1 volume 3 M sodiumacetate and 2 volumes ice cold 100% ethanol. DNA was allowed toprecipitate at -20° C. for >1 hour, then centrifuged for 20 min at13,000 rpm. The DNA pellet was dried under vacuum and resuspended in 100μl TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA).

No detrimental effect was seen with DNA exposed to conditions of 80° C.or 100° C. and 400 atm for exposures of 20 minutes or less. Bands ofintact plasmid DNA were observed in the control lane as well as thelanes containing plasmid DNA treated with supercritical fluid. Thus, theintegrity of DNA is not compromised upon exposure to supercriticalconditions.

EXAMPLE 5 Recovery of DNA From Supercritical Fluid Treated Cells

DNA recovered from the five bacterial species was subjected to agarosegel electrophoresis and ethidium bromide staining. The stained gelsshowed bands of high molecular weight DNA with large quantities of lowmolecular weight nucleic acids present after exposure to supercriticalconditions. RNase digestion of the samples followed by agarose gelelectrophoresis revealed that the low molecular weight nucleic acid wasexclusively RNA. Thus, the DNA recovered after exposure to supercriticalfluid isolation was high molecular weight and had not been extensivelydegraded during the extraction process.

For comparison, DNA was also recovered from each bacterial species bytraditional methods. For control purposes, genomic DNA was extractedfrom filtered and pelleted samples of each bacterial strain using theprocedure of Ausubel et al. (Current Protocols in Molecular Biology,John Wiley & Sons, Inc., New York, 1987, hereby incorporated byreference). Filters were washed in 567 μl TE buffer. From this point on,the procedure was the same for both sets of controls. Cells wereextracted with 30 μl 10% SDS, 3 μl Proteinase K (20 mg/ml) (Sigma, St.Louis, Mo.). The samples were incubated for 1 hour at 37° C., followedby addition of 100 μl 5 M NaCl and 80 μl CTAB. An equal volume ofchloroform/isoamyl alcohol (24:1) was added to each sample followed bycentrifugation for 5-6 minutes. The upper (aqueous) layer was removedand placed in a fresh tube, leaving the filter behind. The DNA wasfurther purified by phenol-chloroform-isoamyl alcohol (24:24:1)extraction followed by precipitation with ethanol and sodium acetate aspreviously described.

EXAMPLE 6 PCR Analysis After Supercritical Fluid Extraction

Universal primers which hybridize to 16s rRNA/DNA of most eubacteriawere synthesized using a DNA synthesizer and purified using theUltrafast cleavage and deprotection kit. Primers were resuspended in TEat a concentration of 1 μg/ml. The 27f primer had the sequence5'-AGAGTTTGATC(C/A)TGGCTCAG-3' (SEQ ID NO: 1) and the 1525r primer hadthe sequence 5'-AAGGAGGTG(A/T)TCCA(A/G)CC-3' (SEQ ID NO: 2). PCRconditions for amplification of the approximately 1.5 kb rDNA sequencewere as follows: 5 minutes initial denaturation at 100° C.; 38 cycles at94° C. for 1 minute (denaturation); 55° C. for 1 minute (cycling); 72°C. for 2 minutes (extension); then 72° C. for 10 minutes (finalextension). Reactions were run in a total volume of 50 μl containing0.05 U/μl Amplitaq DNA polymerase, 0.02 mM dNTPs, 10 μl PCR buffer (75mM Tris-HCl, pH 9.5, 75 mM (NH₄)₂ SO₄, 10 mM MgCl₂ for 5×), and 2 μl ofeach primer. Amplification of the 16s rRNA target sequence showedpositive results with all five bacterial species without requiringfurther purification of DNA.

EXAMPLE 7 DNA hybridization after Supercritical Fluid Treatment

A universal 16s rRNA/DNA 15-mer oligonucleotide probe(5'-ACGGGCGGTGTGT(A/G)C-3') (SEQ ID NO: 3) was end-labeled with ³² Pusing T4 kinase. Sample DNA aliquots and 16s DNA standards were preparedin 0.4 M NaOH (final volume 0.5 ml) and boiled for 10 min. Samples andstandards were blotted onto Biotrans™ nylon membrane (ICN, Irvine,Calif.) using a slot blot apparatus (Bio-Rad, Hercules, Calif.). Blotswere rinsed and dried at 80° C. for 1 hour. The blot was prehybridizedin 0.5 M sodium phosphate, pH 7.2, 1 mM EDTA, 7% SDS for 1 hour at 37°C. The ³² P-labeled probe was added to the blot and incubated overnight.The blot was washed four times using a high stringency wash buffer (20mM Tris-HCl, pH 7-8, 10 mM NaCl, 1 mM EDTA, 0.5% SDS, 37° C.). The blotwas dried, exposed to x-ray film and hybridization signals werequantitated by densitometry using a Visage 110 digital imager(Millipore, Ann Arbor, Mich.). Integrated optical densities werecalculated and hybridization signals were quantitated by interpolationfrom a calibration curve.

DNA hybridization analysis showed an increased recovery of DNAcorresponding to increases in temperature and pressure. The conditionswith the highest pressures and temperatures and longest exposure times(100° C., 400 atm, 30 min) produced the greatest yield of DNA comparedto that obtained by standard SDS lysis procedures. Genomic DNA appearsto be completely unaffected by the extremes of temperature and pressureused in supercritical fluid isolation.

Recovery of nucleic acids from Mycobacterium was 56% and increased to61% when the samples were pretreated with CHCl₃. Nucleic acid recoveryfrom Sphingomonas was 6% at 80° C., 400 atm for 30 minutes; however,CHCl₃ pretreatment increased the recovery to 45%, whilechloroform:methanol (1:1) pretreatment resulted in recovery of 78% ofthe DNA compared to that of the SDS-treated bacterial cultures. Thediffering recoveries may be due to differences in cell membranescompared to the other bacterial species (i.e., the presence of mycolicacid in Mycobacterium and sphingoglycolipids in Sphingomonas).

EXAMPLE 8 Recovery of DNA from Protozoa

Samples of Cryptosporidium muris oocysts were prepared by filtrationonto GF/F glass fiber filters. Each filter contained approximately2.63×10⁷ oocysts. The filters were exposed to supercritical conditionsof 400 atm at 100° C. for 30 minutes and the nucleic acids wererecovered and analyzed by agarose gel electrophoresis and subsequentstaining with ethidium bromide. Nucleic acids ranging from high to lowmolecular weight were visible. The nucleic acid extract was alsosubjected to PCR using primers specific to the C. muris 18s rRNA gene:

5'-MGCTCGTAGTTGGATTTCTG-3' (SEQ ID NO: 4) (forward primer)

5'-TAAGGTGCTGAAGGAGTAAGG-3' (SEQ ID NO: 5) (reverse primer)

After an initial denaturation at 94° C. for 5 minutes, samples werereacted in a thermocycler for 38 cycles as follows: denaturation at 94°C. for 1 minute; annealing at 55° C. for 1 minute, and extension at 72°C. for 2 minutes. The final extension at 72° C. was performed for 10minutes. Amplification of the expected 435 base pair target sequence(Johnson et al., Appl. Environ. Microbiol., 61:3849-3855, 1995) wasobserved. These results demonstrate the successful lysis and recovery ofnucleic acids from C. muris and detection of a specific DNA sequenceusing PCR

It should be noted that the present invention is not limited to onlythose embodiments described in the Detailed Description. Any embodimentwhich retains the spirit of the present invention should be consideredto be within its scope. However, the invention is only limited by thescope of the appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 5                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 # 20               TCAG                                                       - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 #   17             C                                                          - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 15 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 #21                TTCT G                                                     - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 #21                TAAG G                                                     __________________________________________________________________________

What is claimed is:
 1. An apparatus for isolating and extracting nucleicacid from microorganisms within an environmental sample, comprising:asampling cartridge containing said sample, said cartridge having aninput end and an output end; a collection cartridge connected to thesampling cartridge at the output end, said collection cartridge havingan input end and an output end, wherein the collection and samplingcartridges are surrounded by a high pressure compartment within atemperature- and pressure-controlled zone; a high pressure interfaceengaging the input end of the sampling cartridge and the output end ofthe collection cartridge with seals able to withstand high pressure; aplurality of pumps which deliver fluids to the sampling cartridge,wherein at least one of said pumps is a high pressure pump connected toa source of supercritical fluid and at least one of said pumps is aliquid buffer pump connected to a source of aqueous buffer; and acontroller electrically connected to said pumps, such that said pumpssequentially expose the sample to said supercritical fluid and saidaqueous buffer.
 2. The apparatus of claim 1, wherein the nucleic acid isDNA.
 3. The apparatus of claim 1, wherein the nucleic acid is RNA. 4.The apparatus of claim 1, wherein the pumps deliver supercritical fluid,organic solvents, and/or aqueous buffer.
 5. The apparatus of claim 1,wherein the sampling and collection cartridges are disposable.
 6. Theapparatus of claim 1, wherein the sampling and collection cartridges aremade of plastic.
 7. The apparatus of claim 1, wherein the pressure- andtemperature-controlled zone comprises an aluminum block.